Consistent with what was reported for
cultured hippocampal neurons, the YFP signal in multipolar RGCs demonstrated an oscillatory behavior, where signal accumulation was seen traveling between different neurites and areas within the cell ( Figures 1B and INCB024360 solubility dmso 1C, Movie S1, available online). The YFP signal eventually stabilized in a single neurite, which extended to form the axon. From these data we conclude that this construct behaves identically in cultured RGCs and hippocampal neurons, and cultured RGCs progress through a bona fide Stage 2 phase during polarization. We next analyzed how this construct behaves in RGCs polarizing in vivo. Injection of in vitro synthesized Kif5c560-YFP RNA resulted in homogeneous expression in all cells. It was immediately evident that Kif5c560-YFP accumulates basally in the retinal neuroepithelium, even before neurogenesis Dolutegravir begins, resulting in a ring of YFP signal surrounding the lens (Figure 2A). To assess the localization and dynamics of Kif5c560-YFP at the single-cell level, we used a transplantation approach to create mosaic embryos (Figure 2B). ath5:GAP-RFP transgenic
embryos were used because all RGCs are brightly labeled through ath5-regulated fluorescent protein expression, and RGCs can be imaged from before their birth through polarization and axon extension ( Poggi et al., 2005). These embryos were injected with Kif5c560-YFP RNA and P53 morpholino at the one-cell stage, and blastomeres were transplanted into unlabeled host embryos, generating mosaic embryos, where individual cell behaviors could be tracked by time-lapse confocal microscopy. Using this strategy it was apparent that the Kif5c560-YFP construct accumulates basally in all neuroepithelial cells during interphase, being mostly confined to the basal processes. During mitosis and cytokinesis, however, diffuse labeling in the cell body was seen ( Figure 2C). The lack of spindle microtubule
labeling during mitosis Rutecarpine is consistent with the idea that Kif5c560 recognizes stabilized microtubules, and will not label the dynamic spindle microtubules. Consistent with the in vitro data, imaging of RGC axons extending within the eye demonstrated that Kif5c560-YFP accumulates specifically in the growth cone (Figures 2D–2F, Movie S2). Unlike what happens in vitro, however, we found that Kif5c560-YFP accumulation was highly directed in polarizing RGCs in vivo. At the end of the final mitosis marking the birth of RGCs, when RFP signal begins to increase in neonatal RGCs, Kif5c560-YFP is still mainly in the cell body (Figure 2D). Very soon after this, however, a Kif5c560-YFP-positive basal process extends from the cell body. The YFP signal spans a large portion of the re-extending basal process at this time (red arrowheads, Figures 2D and 2F, Movie S2).